The twenty first century is said to be an era of life science. In the life science, proteins have attracted much attention in the post-genome generation and it is important to clear the three-dimensional structures of the proteins with an atom resolution level in order to clarify the correlations between the structure and function of proteins.
In particular, proteins such as enzymes and receptors are main targets of medicines, so that the information on the three-dimensional structures of proteins is important for drug design in the development of medicines.
One of the most frequently used method for analyzing the three-dimensional structure of proteins is X-ray crystal structure analysis. To perform this, a high-quality single crystal must be made.
Use of a crystal of a higher quality without lattice defects enables analyses with a higher precision. At present, however, it is difficult to obtain a high-quality single crystal of a protein. This is a bottle neck in the structure analysis of proteins.
In particular, it is very important to measure the solubility of a protein, which is a basic data for producing protein crystals. In actuality, however, the measurement of solubility of proteins has scarcely been performed since much sample is necessary to do so and it takes a few weeks to a few months to obtain results.
In recent years, methods that allow measurement of solubilities using a relative small amount of sample in a short time have been developed. Such methods include, for example, an optical interference method involving use of a two-beam interferometer (cf., Non-patent documents 1 to 3), a scintillation method, and a microcolumn method.
However, these methods require proteins in an amount of 100 mg or more, even if they are a little amount, by finally getting solubility curves.
<Technology for Producing Protein Crystals>
At present, typical examples of the technology for producing protein crystals include a vapor diffusion method that involves use of the concentration of a protein and the concentration of a precipitating agent as crystallization parameters and a dialysis method that involves use of the concentration of a precipitating agent as a crystallization parameter.
(Vapor Diffusion Method)
This is a method of producing protein crystals by setting a high-concentration protein solution to which a precipitating agent is added, in the form of small droplets in a sealed vessel, filling a salt solution adjusted to a predetermined concentration, which defines the evaporation rate of water, usually, the concentration of which is approximately double the concentration of the precipitating agent in the initial protein solution, and gradually evaporating water from the droplets of the protein solution to precipitate and grow protein crystals in the droplets.
Known methods of this type include a sitting drop method and a hanging drop method. Both the methods have defects. For example, it is difficult to control the amount of water evaporated from the droplets and in addition oxidation denaturation of proteins tends to proceed due to contact of the protein solution with a layer of air.
(Dialysis Method)
This is a method using a semipermeable membrane. Specific examples thereof include a method using a button-shaped cell having small chambers (hereafter, also referred to as “microcell”) and a semipermeable membrane. This is a method of producing protein crystals by charging a small amount, for example, 5 μl of a protein solution adjusted its concentration (containing a precipitating agent in advance if desired) into the small chambers of the cell, sealing the openings of the small chambers with a semipermeable membrane, and immersing the cell in a salt solution with a high concentration to gradually increase the salt concentration in the protein solution in the small chambers by utilizing the phenomenon of dialysis diffusion, thereby precipitating and growing protein crystals in the small chambers.
With the above-mentioned methods, once the apparatus is set, it is only necessary to wait until crystals are generated and grown, it is thus difficult to control the growth of the crystals positively.
Besides, the known methods for producing protein crystals include a batch method that involves preparing a protein solution with a supersaturated state from the beginning and allowing the protein solution to stand to anticipate to generate and grow crystals and a liquid-liquid diffusion method that involves mixing a protein solution and a salt solution by diffusion of solutes to produce crystals. However, it is difficult for the methods that have been ever developed to control the degree of supersaturation that is important for preparing high-quality protein crystals.
<Document Name>
    Non-patent document 1: G. Sazaki, et al., J. Crystal Growth, 169(1996)355    Non-patent document 2: G. Sazaki, et al., J. Crystal Growth, 169(1999)204    Non-patent document 3: K. Ninomiya, et al., J. Crystal Growth, 222(2001)311